Immobilized Metal Affinity Chromatography of Phosphorylated Proteins Using High Performance Sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe³⁺, Ga³⁺, and UO₂ ²⁺ ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe³⁺ and Ga³⁺ ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga³⁺ and Fe³⁺ ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.     

Determination of uric acid in plasma and allantoic fluid of chicken embryos by capillary electrophoresis

Capillary electrophoresis with diode array detection (DAD) was used to determine uric acid (UA) in chicken plasma and the allantoic fluid of chicken embryos. Complete separation of uric and ascorbic acids was attained in less than 10 min in the optimized BGE containing 60 mM MES + 30 mM Tris + 0.001% (w/v) polybrene (pH 6.1). The limit of UA detection (0.2 mg/L) was found to be low enough for sensitive analysis of native plasma and allantoic fluid samples. Range of linearity (1-200 mg/L), repeatability for peak area (CV <4.1%) and migration time (CV < 2.5%), as well as recovery of UA from biological samples (97-100%), were found to be satisfactory. The method was applied to detect the elevated UA concentrations (hyperuricemia) in chicken embryos with induced unilateral renal agenesis. CE/DAD analysis of the chicken plasma can be carried out with a relatively small volume of samples (1 microL).     

Co-isolation of deoxynivalenol and zearalenone with sol–gel immunoaffinity columns for their determination in wheat and wheat products

The paper describes a sample clean-up method for the co-isolation of deoxynivalenol (DON) and zearalenone (ZON), two mycotoxins naturally co-occurring in wheat. The method is based on immunoaffinity columns prepared by co-immobilising anti-DON and anti-ZON antibodies in a porous sol–gel glass. The main task in developing the method consisted in finding a loading medium allowing retention of both analytes as well as a common elution medium for the dissociation of both antigen–antibody complexes formed. This can be achieved by co-extracting DON and ZON with ACN–water (60:40, v/v), reducing the acetonitril concentration to 2.5% before loading an aliquot of the diluted sample extract onto the DON/ZON column. The columns are washed with 5 ml of MeOH–water (10:90, v/v) before DON and ZON are co-eluted with 4 ml of ACN–water (50:50, v/v). Concentrations of DON and ZON are determined with HPLC-UV and HPLC-fluorescence detection, respectively. The sample clean-up method was shown to be applicable to wheat and wheat products, e.g., cornflakes, milk wheat mash and rusk. Spiking experiments (spike level 500 μg DON/kg and 50 μg ZON/kg) resulted in recovery rates from 82% to 111%.     

Application of heptapeptides containing D-amino acid residues immobilized to magnetic particles and Sepharose for the study of binding properties of gastric aspartic proteases

Synthetic heptapeptides containing D-amino acid residues and differing in the content of L-phenylalanine and L-tyrosine residues and their position (Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu, Val-D-Leu-Pro-Phe-Tyr-Val-D-Leu) were immobilized to two types of carriers: glyoxal-activated magnetic agarose particles and CNBr-activated Sepharose. In both cases, peptides were immobilized via their terminal amino group. Immobilized peptides were used for the study of binding properties of two gastric aspartic proteases (porcine pepsin A and rat pepsin C). Porcine pepsin A was adsorbed to all studied peptide-modified magnetic carriers, while rat pepsin C interacted with immobilized ligands only slightly. Similar results were obtained in affinity chromatographic experiments using heptapeptides immobilized to Sepharose.     

Do polysaccharides such as dextran and their monomers really increase the surface tension of water?

It has been reported in the literature that sugars such as dextrose and sucrose increase the surface tension of water. The effect was interpreted as a depletion of the solute molecules from the water-air interface. This paper presents accurate measurements of the surface tension of different concentrations of dextrose solution as well as its polymer (i.e., dextran). An automated drop shape technique called axisymmetric drop shape analysis (ADSA) was used for the surface tension determination. The surface tension measurement is presented as a function of a shape parameter, P(s), which has been used to quantify the range of the applicability of ADSA. The results of the above study show that dextrose solutions decrease the surface tension of water in contradiction to the results obtained from the weight drop method in the literature. The surface tension decreases continuously with increasing concentration. A similar effect was observed for the dextran solutions. To verify that the setup and the methodology are capable of accurately measuring increases in surface tension, a similar experiment was conducted with a sodium chloride solution with a concentration of 1 M. It is well-known that electrolyte solutions, e.g., sodium chloride, increase the surface tension of water. The results obtained from ADSA verify that the sodium chloride increases the surface tension of water by 1.6 mJ/m(2). It is concluded that dextrose and dextran decrease the surface tension of water. Thus, there is no evidence of depletion. To identify the sources of discrepancy between the results of ADSA and those reported in the literature, the experiments were repeated for different concentrations and the rate of drop formation using the drop weight method. It was found that the rate of drop formation is most likely the source of error in the results reported in the literature.     

Magnetic IDA-modified hydrophilic methacrylate-based polymer microspheres for IMAC protein separation

Preparation of a new type of magnetic non-porous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres with hydrophilic properties containing coupled iminodiacetic acid (IDA) is described. The prepared microspheres were used for the immobilization of Ni(II) or Fe(III) ions to show their application in protein binding studies. Human IgG was bound to magnetic Ni(II)-IDA-modified microspheres and conditions of its adsorption and elution were optimized. Non-specific binding of the protein to magnetic microspheres in the absence of Ni(II) ions was low. Fe(III) ions immobilized on magnetic IDA-modified microspheres were used for the specific binding of porcine pepsin, as a model phosphoprotein. The ability of phosphate buffer to release the adsorbed enzyme from the microspheres and a low adsorption of the dephosphorylated protein indicate the participation of phosphate groups in the pepsin interaction. The elaborated method represents a rapid technique that can be used not only for the separation of proteins but also for analytical purposes.     

Low Temperature Aqueous Living/Controlled (RAFT) Polymerization of Carboxybetaine Methacrylamide up to High Molecular Weights

Among the class of zwitterionic polymers poly(carboxybetaine)s (poly(CB)s) are unique, emerging as the only ultra‐low fouling materials known allowing the preparation of biosensors, fouling resistant nanoparticles, and non‐adhesive surfaces for bacteria. Poly(carboxybetaine methacrylate) and poly(carboxybetaine acrylamide) have been prepared via atom transfer radical polymerization (ATRP), however a polymerization with living characteristics has not been achieved yet. Herein, the first successful living/controlled reversible addition fragmentation transfer (RAFT) polymerization of (3‐methacryloylamino‐propyl)‐(2‐carboxy‐ethyl)‐dimethyl‐ammonium (carboxybetaine methacrylamide) (CBMAA‐3) in acetate buffer (pH 5.2) at 70 and 37 °C is reported. The polymerization afforded very high molecular weight polymers (determined by absolute size exclusion chromatography, close to 250 000 g · mol⁻¹ in less than 6 h) with low PDI (<1.3) at 70 °C. The polymerization was additionally carried out at 37 °C allowing to achieve yet lower PDIs (1.06 ≤ PDI ≤ 1.15) even at 90% conversion, demonstrating the suitability of the polymerization conditions for bioconjugate grafting. The living character of the polymerization is additionally evidenced by chain extending poly(CBMAA‐3) at 70 and 37 °C. Block copolymerization from biologically relevant poly[N‐(2‐hydroxypropyl)methacrylamide] macroCTAs was additionally performed.

     

Poly(ethylene glycol) enhances the surface activity of a pulmonary surfactant

The primary role of lung surfactant is to reduce surface tension at the air–liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3350; 8000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m². PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy.     

Iodine in mineral waters: A comparison of results from two analytical techniques

A comparative study of the determination of iodine in mineral waters is presented. Iodine was first determined by a standard titrimetric method and the results obtained were compared with those obtained by the faster and more sensitive radiochemical neutron activation method. For a series of waters, the results obtained by the two techniques were in fair agreement for the higher concentration levels of iodine, but for very low iodine levels the titrimetric method was insufficiently sensitive. The RNAA procedure was checked by standard addition experiments, and shown to be also valid when iodine was present as iodate.The paper was presented in part at the ISM XI, Wiesbaden, August 1989     

Lattices and Quantum Logics with Separated Intervals, Atomicity

It is well known that a Boolean algebra B isatomic (atomistic) iff the interval topology on B isHausdorff. But this no longer holds for orthomodularlattices (quantum logics). There exist (even complete) atomic orthomodular lattices the intervaltopology of which is not Hausdorff. We show that anothercharacterization of atomicity for Boolean algebras isthe following: A Boolean algebra B is atomic iff B has separated intervals. Furthermore, we showthat the interval topology on a complete orthomodularlattice L is Hausdorff iff L has separated intervals iffL is atomic and it has separated intervals. An orthomodular lattice L with orthomodularMacNeille completion has separatedintervals iff L is atomic and it has separated intervalsiff the interval topology on isHausdorff.